PROJECT SUMMARY A long-standing technical challenge in neuroscience is high-resolution functional and molecular imaging of the awake mouse brain. The need is evident and pressing, because anesthesia can significantly reduce the overall brain activity and alter multiple forms of brain dynamics. The profound effects of anesthesia may confound the readouts of conventional microscopies, which require preparations of anesthetized animals, and thus impose significant limitations on the interpretation and translation of basic neuroscience findings. Moreover, incapable of imaging the awake brain for direct comparison with the anesthetized counterpart, conventional microscopies are of very limited utility in examining the important yet elusive roles of general anesthesia in the progression of multiple life-threatening brain disorders (e.g., ischemic stroke and Alzheimer's disease, which are the leading causes of death and disability in the United States). In addressing this challenge, recent efforts have extended the scope of fluorescence microscopy to the awake brain. While this molecular imaging technology advances and rapidly expands our understanding of the neural activities underlying behavior, high-resolution functional imaging of the coevolving hemodynamics falls far behind. This project aims to bridge the increasing technology gap by developing a first-of-a-kind photoacoustic microscopy (PAM) instrumentation for functional imaging of cerebral hemodynamics and metabolism at high spatiotemporal resolution in awake mice. The unprecedented speed of the proposed awake-brain PAM (1 MHz A-line rate), enabled by the innovative designs of wide-field optical-mechanical hybrid scan and MHz-repetition-rate dual-wavelength Raman laser, will exceed that of the existing multi-parametric PAM by two orders of magnitude and will enable spatiotemporal visualization of the functional and metabolic responses of the brain to neural stimulations and disease onsets without the influence of anesthesia. The complementary algorithms for statistical, spectral and correlation analysis of the same PAM dataset will further push the technology envelope by enabling simultaneous and comprehensive quantification of the total concentration and oxygen saturation of hemoglobin, blood flow and perfusion, and metabolic supply and demand at the microscopic level. This technology innovation will open up new and exciting opportunities in basic and translational neuroscience, including the mechanistic study of anesthetic neuroprotection in ischemic stroke proposed in this project. In turn, this stroke study will provide an ideal setting to assess the potential of awake-brain MHz-PAM in the context of a clinically important brain disease and pave the way for future studies of neurovascular coupling and neuromodulation in the awake brain. These efforts, together, hold the potential to establish PAM as a new enabling technology in brain research.